In response to RFA-AG-06-003, this application proposes to investigate mechanisms that link the adipogenic phenotype of aging muscle to the development of insulin resistance. Based on our preliminary data, we hypothesize that increased perimuscular adipogenesis contributes to insulin resistance via mechanisms that link directly to fatty acid-induced mitochondrial stress. We have therefore proposed studies to determine how lipid deposits within and between skeletal myofibers impact mitochondrial performance, insulin sensitivity and the interplay between these two functional endpoints. We further hypothesize that habitual exercise combats the adverse effects of lipid infiltration by remodeling muscle mitochondria in manner that enables these organelles to better cope with a high lipid load. These hypotheses will be tested using a two-pronged approach that applies comprehensive mass-spectrometry (MS) based metabolic profiling strategies to both in vitro and in vivo studies of human muscle. First, our plan will employ a newly developed human adipocyte-myocyte co-culture system to investigate i) the impact of adipocytes on the metabolic function of neighboring myocytes;and ii) the underlying mechanisms that link increasing adipogenesis to impaired insulin action. We predict that lipid-induced mitochondrial stress will emerge as a primary event that connects adipogenic burden to impaired glucose homeostasis, and moreover, that this event might be exacerbated in myocytes from aged compared to young donors. Second, using existing data/specimens from the STRRIDE study we will translate findings from our in vitro model to human physiology. To this end, we will examine how lipid infiltration of muscle (both intramuscular and perimuscular) relates to both insulin sensitivity and metabolic/transcriptional markers of mitochondrial performance, in the context of an exercise training intervention. These goals will be accomplished by a multidisciplinary research team from the Duke University Stedman Nutrition and Metabolism Center. Completion of the aims of this study will not only provide mechanistic information about the relationship between lipid stores in and around skeletal muscle and whole body insulin action - a major determinant of morbidity related to obesity, diabetes and aging - but will also provide insight into the most efficacious exercise prescription for preventing and correcting deficient skeletal muscle insulin action in these conditions.